Testis is the one and only location of spermatogenesis and sexual hormone production. Spermatogenesis is a complicated physiological process regulated by many genes specifically and differentially expressed in the testis. In this study, Transmembrane Protein 225 (TMEM225), which is specifically expressed in rat testis, has been identified. TMEM225 was cloned from the testis cDNA library and was mapped to chromosome 8q22 by browsing the University of California Santa Cruz genomic database. It contains an open reading frame with a length of 696 bp, encoding a protein with four putative transmembrane helices. TMEM225 mRNA expression was evaluated by reverse transcription-polymerase chain reaction and in situ hybridization. In addition, the subcellular location of TMEM225 was evaluated. The results obtained highlighted age related specific expression of TMEM225 in testis, specifically during the adult period after age of 13 months. In situ hybridization analysis indicated that TMEM225 mRNA was mainly expressed in spermatocyte cells and round spermatids. Green fluorescence protein localization analysis showed that rat TMEM225 mainly surrounded the nuclear membrane, with a minority distribution in the cytoplasm, and the distribution of TMEM225 was affected by the deletion of N-terminal transmembrane domain. As the expression phase is not related to the first wave of spermatozoon development, our data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis.